Alkylated hormone-releasing peptides and method of treatig mammals therewith

ABSTRACT

This invention provides novel growth hormone-releasing peptides and a method for increasing the release and raising the levels of growth hormone in mammals. The invention also provides a method for increasing the growth rate of meat producing animals, treating the symptoms of growth hormone deficiencies in mammals and improving the efficiency of feed utilization by meat producing and dairy animals.

The present invention was supported in part by an award from theNational Institute of Health, Grant No. DK 30167. The U.S. Governmenthas certain rights in this invention.

This application is a continuation-in-part of application Ser. No.07/048,203, filed May 11, 1987, abandoned.

BACKGROUND OF THE INVENTION

During the past decade growth hormone-releasing factors (GRF) of humanpancreatic islet tumor origin (hpGRF) have been isolated, characterizedand shown to possess growth hormone (GH)-releasing activity in ratanterior pituitary in vitro and in vivo by (1) R. Guillemin, P. Grazeau,P. Bohlen, F. Esch, N. Ling, and W. B. Wehrenberg [Science, 218, 585(1982)]and (2) J. Spiess, J. Rivier, M. Thorner, and W. Vale[Biochemistry, 21, 6037 (1982)]. A synthetic hp GRF(1-29)-NH₂, anamidated fragment of the natural hp GRF, has also been prepared andreported to possess full intrinsic biological activity by Spiess et al.of reference (2).

Additionally, it has been found that an increase in growth hormone(GH)-release in animals can be attained by substituting D-amino acidsfor L-amino acids of natural hpGRF, especially in the 2 and 3 positions,(Lance et al., Biochemical and Biophysical Research Communications. Vol.119, No. 1, 1984, pp 265-272: Vale, Jr. et al., U.S. Pat. No.4,528,190). Likewise, Vale, Jr. et al., have disclosed in theabove-mentioned patent that an increase in GH-release is obtained byinserting an N .sup.α -methyl (or C .sup.α -methyl-substituted aminoacid in positions 1 and 2 of hpGRF. These investigators, however,neither extended the length of the alkyl group beyond methyl on theN-terminus of the hpGRF peptides, nor suggested that such extensionwould enhance the activity of the hpGRF peptides.

SUMMARY OF THE INVENTION

This invention relates to novel growth hormone-releasing peptides andthe use thereof for enhancing the release and increasing the growthhormone levels in mammals.

Unexpectedly, we have now discovered that elongation of the alkylsubstituent at the N-terminus and concomitant alkylation of the basicamino acids within the peptide chain, such as lysine in positions 12 and21, results in substantial increase in potency in eliciting GH-releaseover that stimulated by the natural hpGRF(1-29)-NH₂, hpGRF(1-44)NH₂ or[N α-MeTyr¹ ]hpGRF(1-29)-NH₂.

The present invention thus provides novel mono-and per-alkylatedpeptides, that are derived by reductive alkylations of GH-releasingpeptides through the use of sodium cyanoborohydride and aldehydes orketones. These peptides of the present invention are extremely potent instimulating GH-release in warm-blooded animals, including humans.

The novel peptides of this invention are defined as any peptide havingfrom 29 to 44 amino acid residues sequenced as shown in formula A below,and terminated at any amino acid residue function between the 29th and44th acid residue positions, with the proviso, that the carboxyl moietyof the amino acid residue at the C-terminus is provided with an R₁₇function; wherein R₁₇ is OH, OR₁₈, NH₂ or NHR₁₈ and R₁₈ is C₁ -C₉ alkyl(straight or branched). C₃ -C₆ cycloalkyl, benzyl, naphthyl, methylnaphthyl or mono-substituted benzyl wherein the substitution is halogen,methoxy, nitro or cyano; with the proviso that when the peptide is therat GRF, R₁₅ is Asn and R₁₆ can only be OH, NH₂, OR₁₈ or Gly; and whenGly is the carboxy-terminus amino acid, the functional group is an acidOH.

The invention also relates to the pharmaceutically acceptable salts ofthe formula (A) peptides.

The novel peptides of the invention are illustrated by formula A asfollows: ##STR1## wherein R₁ is N.sup.α -R₁₈ -Tyr, N.sup.α -R₁₈ -His,N.sup.α -R₁₈ -Phe, N.sup.α -R₁₈ -O-Tyr, N.sup.α -R₁₈ -D-His, or N.sup.α-R₁₈ -D-Phe;

A is Ala, D-Ala or N-Me-D-Ala;

B is Asp, D-Asp;

R₂ is Asn, D-Asn or Ser;

E is Tyr, D-Tyr;

R is Arg, Lys or N.sup.ε -R₁₈ -Lys;

R₄ is Ile or Val;

R₃ is Gly, α-aminoisobutyric, α-aminobutyric, Ala, Gln, Asn, Leu, Ile orVal;

R₅ is Ser or tyr;

R₆ is His or Gln;

R₇ is Glu or Asp;

I is Met, Leu, Nle, Ile, Val, nor Val, Ser, thr, Asn or Gln;

R₈ is Ser or Asn;

R₉ is Ser or Arg;

R₁₀ is Arg or Gln;

R₁₁ is Arg or Gly;

R₁₂ is Ala or Ser;

R₁₃ is Arg or N.sup.ε -R-Lys;

R₁₄ is Phe, Val or Ala;

R₁₅ is Asn or Arg;

R₁₆ is Gly or Leu;

R₁₇ is OH, OR₁₈, NH₂ NHR₁₈ ;

R₁₈ is C₁ -C₉ alkyl (straight or branched). C₃ -C₆ cycloalkyl, benzyl,naphthyl, methyl naphthyl or mono-substituted benzyl wherein thesubstitution is halogen, methoxy, nitro or cyano; with the proviso thatwhen the peptide is the rat GRF, R₁₅ is Asn and R₁₆ can only be OH, NH₂,OR₁₈ or Gly; and when Gly is the carboxy-terminus amino acid, thefunctional group is an acid OH; with the further proviso that theformula A peptide can be terminated at any amino acid residue betweenthe 29th and 44th acid residue positions provided that the carboxylmoiety of the amino acid residue at the C-terminus is provided with anR₁₇ function, as described above; and the pharmaceutically acceptablesalts thereof.

Preferred GRF peptides of the invention are defined in formulas I, IIIand III as follows: ##STR2##

Other preferred GRF peptides of this invention are ##STR3##

Other growth hormone releasing peptides of the present invention includethe per-alkylated particularly the per-isopropylated, peptides forhumans, swine, cattle, goats, sheep and rats. These peptides may becharacterized as follows: ##STR4##

In keeping with standard nomenclature, the abbreviations used above forchiral amino acid residues and used throughout the present specificationand claims are as follows:

Code for L and D-Amino Acids, per IUPAC-IUB Commission on BiochemicalNomenclature: Biochemistry 11, 1726-1732 (1972)

    ______________________________________                                        His = Histidine      Val = Valine                                             Ser = Serine         Phe = Phenylalanine                                      Asp = Aspartic Acid  Thr = Threonine                                          Ala = Alanine        Asn = Asparagine                                         Tyr = Tyrosine       Gln = Glutamine                                          Arg = Arginine       Met = Methionine                                         Leu = Leucine        Ile = Isoleucine                                         Lys = Lysine         Glu = Glutamic Acid                                      Nle = Norleucine     NorVal = norValine                                       sd,5 .sub.-- D-Ala = sd,5 .sub.-- D-Alanine                                   sd,5 .sub.-- D-Asp = sd,5 .sub.-- D-Aspartic Acid                             sd,5 .sub.-- D-Ser = sd,5 .sub.-- D-Serine                                    sd,5 .sub.-- D-Tyr = sd,5 .sub.-- D-Tyrosine                                  sd,5 .sub.-- D-His = sd,5 .sub.-- D-Histidine                                 sd,5 .sub.-- D-Phe = sd,5 .sub.-- D-Phenylanine                               N-Me-sd,5 .sub.-- D-Ala = N-methyl-sd,5 .sub.--  D-alanine                    sd,5 .sub.-- D-Asn = sd,5 .sub.-- D-Asparagine                                ______________________________________                                    

Additionally, it should be noted that unless otherwise specified, theamino acid residues that are named herein without the prefix L will, infact, refer to the naturally occurring absolute configuration L.

Other abbreviations used in the present specification are

    ______________________________________                                        Fmoc = fluorenylmethyloxycarbonyl                                             Boc =  .sub.- t-butyloxycarbonyl                                              Tos =  -p-toluenesulfonyl                                                     hplc = high performance liquid chromatography                                 tlc = thin-layer chromatography                                               TFA = trifluoroacetic acid                                                    Ac = acetyl                                                                   Z = benzyloxycarbonyl                                                         ______________________________________                                    

The term "pharmaceutically acceptable salts" as used herein, refers tonon-toxic alkali metal, alkaline earth metal, ammonium, organoammoniumand metallic salts commonly used in the pharmaceutical industry. Thesesalts include, but are not limited to, the sodium, potassium, lithium,calcium, magnesium, zinc, ammonium, and trimethylammonium salts whichare prepared by methods well known art. The term also includes non-toxicacid addition salts such as hydrochloride, hydrobromide, acetate,phosphate, sulfate, citrate, laurate, stearate, palmoate, and oleate,but are not limited to them. These acid addition salts are also preparedby methods well known in the art.

Further, the term "organoammonium" is defined as a group consisting of apositively charged nitrogen atom joined to from one to four aliphaticgroups, each containing from one to 20 carbon atoms. Among the organicammonium groups which are illustrative for the preparation of thealiphatic ammonium salts of this invention are: monoalkylammonium,dialkylammonium, trialkylammonium, tetraalkylammonium,monoalkenylammonium, dialkenylammonium, trialkenylammonium,monoalkynylammonium, dialkynylammonium, trialkanolammonium, C₅ -C₆cycloalkylammonium, piperidinium, morpholinium, pyrrolidinium,benzylammonium, and equivalents thereof.

Solid-phase synthesis of the peptides of the present invention can becarried out on a Beckman 990 automatic peptide synthesizer. PreparativeHPLC can be performed on a thick-walled glass column (2.5×45 cm)containing Whatman LRP-1 reverse phase packing (C₁₈ silica 13-22 μm)pumped with Fluid Metering Company pump and pulse damper. Amino acidanalyses can be run on a Beckman 119 CL analyzer and processed with aSystem AA computing integrator.

Amino acid derivatives utilized in the preparation of the compounds ofthe present invention are available from several chemical supply housesincluding: Bachem, Inc., Torrance, Calif., and Chemical Dyanamics, Inc.,Plainfield, N.J.

The peptides having configurations shown as formulas A, I, II or III,can be conveniently prepared by standard solid-phase techniques; forexample, the C-terminal protected amino acid can be attached to achloromethyl resin, a hydroxymethyl resin, a benzhydrylamine (BHA) resinor a p-methylbenzylhydrylamine (p-Me-BHA) resin. One such chloromethylresin is sold under the trade name Bio-Beads SX-1 by Bio RadLaboratories, Richmond, Calif. The preparation of the hydroxymethylresin is described by Bodansky et al , Chem. Ind. (London) 38. 1597(1966). The BHA resin has been described by Pietta and Marshall, Chem.Common. 650 (1970) and Commercially available from Bachem, Inc.,Torrance, Calif.

According to one embodiment of the invention, the peptides of formulasA, (I), (II) and (III), are prepared by means of solid-phase peptidesynthesis by standard procedures, although it may also be prepared bytreatment of the peptide-resin with ammonia to give the desiredside-chain protected amide or with an alkylamine to give a side-chainprotected alkylamide or dialkylamide. Further, the asp and glu wouldalso be derivatized residues present as benzyl esters.

The α-amino protecting group is Fmoc for the amino acid in position one,and the side-chain protecting group is Boc instead of Z for theappropriate preceding amino acid, when the chloromethyl or hydroxymethylresin is used.

Side-chain protection can then be removed in the usual fashion bytreatment with HF to give the free peptide amides, alkylamides, ordialkylamides.

In preparing the esters of this invention, the resins used to preparethe acids of formulas A, (I), (II) and (III), where R₁₇ is OH, can beemployed, and the side-chain protected peptide can be cleaved with abase and appropriate alcohol, i.e., methanol. Side-chain protectinggroups can then be removed in the usual fashion by treatment with HF toobtain the desired ester. Alkylation at the N-terminus and concomitantalkylation of the basic amino acids with the peptide chain, such aslysine in the positions 12,21 and 41, can be achieved by removing theBOC protecting groups from the N-Terminus and FMOC groups from the basicamino acids with the resin, subjecting the thus prepared peptide resinto a reduction with an alkali metal cyanoborohydride in the presence ofDMF and acetic acid, or in the case of FMOC groups removal with 50%piperidine in DMF, and alkylating the thus prepared resin with theappropriate aldehyde or ketone to yield the desired alkylated GRF resinwhich can then be cleaved with HF to give the alkylated GRF amine.##STR5##

The peptides of the present invention are useful for treatments ofsymptoms related to growth hormone deficiencies. They are also usefulfor increasing wool growth, for increasing the rate of growth ofmeat-producing animals, for improving the carcass quality ofmeat-producing animals (i.e., more protein and less fat), for improvingfeed efficiency in meat-producing animals and dairy cows, and forincreasing milk production in dairy herds.

As indicated, the compounds of the present invention are effective forincreasing the release of growth hormone in mammals, including humans.In practice it is found that said compounds of the invention areeffective when administered to mammals in an amount sufficient toprovide said treated mammals with from 0.000001 to 0.1 mg/kg ofmammalian body weight/day of the formula I or II compounds of thepresent invention.

In order to facilitate a further understanding of the invention, thefollowing examples are presented primarily for the purpose ofillustrating certain more specific details thereof. The invention is notto be deemed limited thereby except as defined in the claims.

EXAMPLE 1

Nle²⁷ -GRF(11-29)-methylbenzhydrylamine resin

Methylbenzhydrylamine-polystyrene resin (Vega Biochemicals, Inc.) (3.80g, 2.0 mmole) in the chloride ion form is placed in the reaction vesselof a Beckman 990B peptide synthesizer programmed to perform thefollowing reaction cycle: (a) methylene chloride; (b) 33%trifluoroacetic acid in methylene chloride (2 times for 1 and 25 mineach); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f)10% triethylamine in chloroform.

The neutralized resin is stirred with Boc-N^(G) -tosyl-Arg (6 mmole) anddiisopropylcarbodiimide (6 mmole) in methylene chloride for 1 hour andthe resulting amino acid resin is then cycled through steps (a) to (g)in the above wash program. The following amino acids (6 mmole) are thencoupled successively by the same procedure (Gln and Asn in the presenceof equimolar 1-hydroxybenzotriazole)):

Boc-O-benzyl-Ser, Boc-Nle, Boc-Ile, Boc-benzyl-Asp-, Boc-Gln, Boc-Leu,Boc-Leu, Boc-N^(e) -2-chlorocarbenzoxy-Lys, Boc-tosyl-Arg, Boc-Ala,Boc-benzyl-Ser, Boc-Leu, Boc-Gln, Boc-Gly, Boc-Leu, Boc-Val,Boc-Cl-Z-Lys, Boc-tosyl-Arg.

After drying, the resin weighs 7.4 g.

EXAMPLE 2 [N.sup.α -Ethyl-Tyr¹, Nle²⁷ ]GRF(1-29)-methylbenzhydrylamineresin

The resin described in Example 1 (2.0 g, 0.5 mmole) is coupledsuccessively with Boc-2-Br-Z-Tyr, Boc-benzyl-Ser, Boc-Asn,Boc-0-benzyl-Thr, Boc-Phe, Boc-Ile-Boc-Ala, Boc-benzyl-Asp, Boc-Ala, andBoc-Tyr. The last Boc group then removed and the resin dried to give2.65 g of material.

Resin (1,32g, 0.25 mmole) was suspended in DMF/1% acetic acid to whichwas added NaBH₃ CN (3 mmole) followed by acetaldehyde (1 ml). After 1hour, the resin was completely negative to the Kaiser ninhydrin test.

EXAMPLE 3

[N.sup.α -Isopropyl-Tyr¹,Nle²⁷ ]GRF(1-29)methylbenzhydrylamine resin

The resin described in Example 1 (2.0 g, 0.5 mmole) is coupledsuccessively with Boc-2-Br-Z-Tyr, Boc-benzyl-Ser, Boc-Asn,Boc-0-benzyl-Thr, Boc-Phe, Boc-Ile-Boc-Ala, Boc-benzyl-Asp, Boc-Ala, andBoc-Tyr. This last Boc group was then removed and the resin dried togive 2.65 g of material.

Resin (1.32g, 0.25 mmole) was suspended in DMF/1% acetic acid to whichwas added NaBH₃ CN (3 mmole) followed by acetone (1 ml). After 1 hour,the resin was completely negative to the Kaiser ninhydrin test.

EXAMPLE 4 GRF(3-29)-methylbenzhydrylamine resin

Methylbenzhydrylamine-polystyrene resin (Vega Biochemicals, Inc.) (5.80g, 3.0 mmole) in the chloride ion form is placed in the reaction vesselof a Beckman 990B peptide synthesizer programmed to perform thefollowing reaction cycle: (a) methylene chloride; (b) 33%trifluoroacetic acid in methylene chloride (2 times for 1 and 25 mineach); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f)10% triethylamine in chloroform.

The neutralized resin is stirred with Boc-N^(G) -tosyl-Arg (9 mmole) anddiisopropylcarbodiimide (9 mmole) in methylene chloride for I hour andthe resulting amino acid resin is then cycled through steps (a) to (g)in the above wash program. The following amino acids (9 mmole) are thencoupled successively by the same procedure (Gln and Asn in the presenceof equimolar 1-hydroxybenzotriazole): Boc-O-benzyl-Ser, Boc-Met,Boc-Ile, Boc-benzyl-Asp, Boc-Gln, Boc-Leu, Boc-Leu, Boc-N^(e)-2-chlorocarbenzoxy-Lys, Boc-tosyl-Arg, Boc-Ala, Boc-benzyl-Ser,Boc-Leu, Boc-Gln, Boc-Gly, Boc-Leu, Boc-Val, Boc-Cl-Z-Lys,Boc-tosyl-Arg, Boc-2-Br-Z-Tyr, Boc-benzyl-Ser, Boc-Asn,Boc-O-benzyl-Thr, Boc-Phe, Boc-Ile-Boc-Ala, Boc-benzyl-Asp.

The dried resin weighed 18.5 g.

EXAMPLE 5 [N.sup.α -Isopropyl-Tyr¹,D-Ala²]GRF(1-29)-methylbenzhydrylamine resin

The resin described in Example 4 (1.5 g, 0.5 mmole) is coupledsuccessively with Boc-D-Ala and Boc-Tyr. The last Boc group is thenremoved and resin was suspended in DMF/1% acetic acid to which was addedNaBH₃ CN (3 mmole) followed by acetone (3 ml). After 18 hours, the resinis completely negative to the Kaiser ninhydrin test.

EXAMPLE 6 [N.sup.α -Isopropyl-Tyr¹ l]GRF(1-29)-methylbenzhydrylamineresin

The resin described in Example 4 (1.5 g, 0.5 mmole) is coupledsuccessively with Boc-D-Ala and Boc-Tyr. The last Boc group is thenremoved and resin was suspended in DMF/1% acetic acid to which was addedNaBH₃ CN (3 mmole) followed by acetone (3 ml). After 18 hours, the resinis completely negative to the Kaiser ninhydrin test.

EXAMPLE 7 [N.sup.α -Cyclohexyl-Tyr¹ ]GRF(1-29)-methylbenzhydrylamineresin

The resin described in Example 4 (1.5 g, 0.5 mmole) is coupledsuccessively with Boc-Ala and Boc-Tyr. The last Boc group is thenremoved and resin was suspended in DMF/1% acetic acid to which was addedNaBH₃ CN (3 mmole) followed by cyclohexanone (3 ml). After 18 hours, theresin is completely negative to the Kaiser ninhydrin test.

EXAMPLE 8 [N.sup.α -Benzyl-Tyr¹ ]GRF(1-29)-methylbenzhydrylamine resin

The resin described in Example 4 (1.6 g, 0.5 mmole) is coupledsuccessively with Boc-Ala and Boc-Tyr. The last Boc group is thenremoved and resin was suspended in DMF/1% acetic acid to which was addedNaBH₃ CN (3 mmole) followed by benzaldehyde (3 ml). After 2 hours, theresin is completely negative to the Kaiser ninhydrin test.

EXAMPLE 9 [N.sup.α -5-nonyl-Tyr¹ ]GRF(1-29)-methylbenzhydrylamine resin

The resin described in Example 4 (1.5 g, 0.5 mmole) is coupledsuccessively with Boc-Ala and Boc-Tyr. The last Boc group is thenremoved and resin was suspended in DMF/1% acetic acid to which was addedNaBH₃ CN (3 mmole) followed by 5-nonanone (3 ml). After 3 days, theresin is very positive to the Kaiser ninhydrin test and is stirred at50° C. overnight whereupon a negative test is obtained.

EXAMPLE 10 [N.sup.α 3-pentyl-Tyr¹ ]GRF(1-29)-methylbenzhydrylamine resin

The resin described in Example 4 (1.6 g, 0.5 mmole) is coupledsuccessively with Boc-Ala and Boc-Tyr. The last Boc group is thenremoved and resin was suspended in DMF/1% acetic acid to which was addedNaBH₃ CN (3 mmole) followed by 3-pentanone (3 ml). After 18 hours, theresin is very positive to the Kaiser ninhydrin test and is stirred at50° C. for 3 hours whereupon a negative test is obtained.

EXAMPLE 11 [N.sup.α -Ethyl-Tyr¹,Nle²⁷ ]GRF(1-29)NH₂

Peptide resin from Example 2 is mixed with anisole (5 ml) and anhydroushydrogen fluoride (35 ml) at 0° C. and stirred for 45 min. Excesshydrogen fluoride is evaporated rapidly under a stream of dry nitrogenand free peptide precipitated and washed with ether. The crude peptideis then dissolved in a small volume of 2 M AcOH and eluted on a column(2.5×100 cm) of Sephadex G-50 in the same buffer. Fractions containing amajor component by uv absorption and thin layer chromatography are thenpooled, evaporated to a small volume and applied to a column (1.5×45 cm)of Vydac octadecylsilane (15 um).

This is eluted with a linear gradient of 15-45% acetonitrile in 0.1%trifluoroacetic acid in water. Fractions are examined by thin layerchromatography and analytical high performance liquid chromatography andpooled to give maximum purity. Repeated lyophilization of the solutionfrom water gives 34 mg of the product as a white, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 12 [N.sup.α -Isopropyl-Tyr¹,Nle²⁷ ]GRF(1-29)NH₂

Peptide resin from Example 3 is cleaved with HF and purified under theconditions described in Example 11 to give 82 mg of the product as awhite, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 13 [N.sup.α -Isopropyl-Tyr¹,D-Ala² ]GRF(1-29)NH₂

Peptide resin from Example 5 is cleaved with HF and purified under theconditions described in Example 11 to give 80 mg of the product as awhite, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 14 [N.sup.α -Isopropyl-Tyr¹ ]GRF(1-29)NH₂

Peptide resin from Example 6 is cleaved with HF and purified under theconditions described in Example 11 to give 170 mg of the product as awhite, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 15 [N.sup.α -Cyclohexyl-Tyr¹ GRF(1-29)NH₂

Peptide resin from Example 7 is cleaved with HF and purified under theconditions described in Example 11 to give 132 mg of the product as awhite, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alylated.

EXAMPLE 16 [N.sup.α -Benzyl-Tyr¹ ]GRF(1-29)NH₂

Peptide resin from Example 8 is cleaved with HF and purified under theconditions described in Example 11 to give 82 mg of the product as awhite, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 17 [N.sup.α -5-nonyl-Tyr¹ ]GRF(1-29)NH₂

Peptide resin from Example 9 is cleaved with HF and purified under theconditions described in Example 11 to give 68 mg of the product as awhite, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 18 [N.sup.α -3-pentyl-Tyr¹ ]GRF(1-29)NH₂

Peptide resin from Example 10 is cleaved with HF and purified under theconditions described in Example 11 to give 143 mg of the product as awhite, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 19 [N.sup.α -isopropyl-Tyr¹, N.sup.ε -isopropyl-Lys¹²,21]GRF(1-29)-NH₂

GRF(1-29)NH₂ (35 mg, 1 umole) is dissolved in DMF/1% AcOH (3 ml) andacetone (0.5 ml). NaBH₃ CN (4 mg) is then added and the mixture stirredfor 4 hours. After removal of the volatile components, the peptide ispurified under the conditions described in Example 11 to give 25 mg ofthe product as a white, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition or the alkylated.

EXAMPLE 20 [N.sup.α -isopropyl-Tyr¹,N .sup.ε -isopropyl-Lys¹²,21]GRF(1-29)OH

GRF(1-29)OH (35 mg, 1 umole) is dissolved in DMF/1% AcOH (3 ml) andacetone (0.5 ml). NaBH₃ CN (4 mg) is then added and the mixture stirredfor 4 hours. After removal of the volatile components, the peptide ispurified under the conditions described in Example 11 to give 28 mg ofthe product as a white, fluffy powder.

The product is found to be homogeneous by hplc and tlc. Amino acidanalysis of an acid hydrolysate and fast atom bombardment massspectrometry confirms the composition of the alkylated.

EXAMPLE 21 Evaluation of peptide effects on growth hormone release inmammals using the rat as the test species

In this evaluation, the procedures described by W. A. Murphy et al.,Endocrinology 109: 491-495 (1980), were employed.

In growth hormone (GH) experiments, male rats (Charles Rivers) wereanesthetized with NEMBUTAL® (6 mg per 100 g body weight) which alsoserved to maintain stimulated plasma GH levels. Exactly 30 minutes afterthe rats were anesthetized, 0.5 mL of saline or the test peptide insaline was administered as a SC bolus. A 1 mL blood sample was drawnfrom the jugular vein 15 minutes after the injection of the peptide insaline or at the indicated times in the time course assay (p26). GHlevels were determined using NIADDKD rat GH RIA components.

    ______________________________________                                        BIOLOGICAL ACTIVITY OF GRF(1-29)NH.sub.2 ANALOGS IN                           THE PENTOBARBITAL-ANESTHETIZED RAT                                                       DOSE                                                               TEST SUB-  (ug/100  PLASMA GH#                                                STANCE     g BW)    (ng/ml)      POTENCY*                                     ______________________________________                                        Saline     --        243 ± 25 (8)                                           .sub.-- N.sup.α MeTry.sup.1                                                       30       1843 ± 196 (6)                                                                          0.42                                          "         75       2613 ± 418 (6)                                                                          (0.30-0.60)                                  Saline     --        246 ± 36 (7)                                           .sub.-- N.sup.α EtTyr.sup.1,Nle.sup.27                                             4       2031 ± 294 (6)                                                                          5.0                                           "         10       3878 ± 288 (6)                                                                          (3.6-7.0)                                    Saline     --        243 ± 25 (8)                                           .sub.-- N.sup.α iPrTyr.sup.1, Nle.sup.27                                           0.12     685 ± 94 (6)                                                                           41                                            "          0.30    1020 ± 208 (6)                                                                          (28-54)                                      Saline     --        325 ± 52 (9)                                           .sub.-- N.sup.α iPrTyr.sup.1                                                       0.2     1334 ± 394 (6)                                                                          .sup.˜ 60                               "          2       3250 ± 362 (6)                                          "         20       3290 ± 241 (6)                                          .sub.-- N.sup.α iPrTyr.sup.1, D-Ala.sup.2                                          0.2     1608 ± 274 (6)                                                                          .sup.˜ 70                               "          2       3836 ± 397 (6)                                          "         20       3099 ± 485 (6)                                         Saline     --        59 ± 9 (9)                                             .sub.-- N.sup.α cyclohexylTyr.sup.1                                                0.2      58 ± 21 (6)                                                                             .sup.˜ 2                               "          2        56 ± 10 (6)                                            "         20        727 ± 144 (6)                                          .sub.-- N.sup.α benzylTyr.sup.1                                                    0.2      213 ± 32 (6)                                                                           .sup.˜ 50                               "          2        606 ± 51 (6)                                           "         20       1063 ± 140 (6)                                         Saline     --        63 ± 8 (9)                                             .sub.-- N.sup.α -5-nonylTyr.sup.1                                                  0.2      46 ± 4 (6)                                                                             <0.5                                          "          2        85 ± 31 (6)                                            "         20        104 ± 12 (6)                                          Saline     --        91 ± 11 (9)                                           GRF(1-29)NH.sub.2                                                                        10        222 ± 50 (4)                                           "         25        622 ± 63 (6)                                           .sub.-- N.sup.α iPrTyr.sup.1,                                                      0.08     308 ± 24 (6)                                          N.sup.ε iPrLys.sup.12,21                                              N.sup.α iPrTyr.sup.1,                                                               0.20     467 ± 86 (6)                                                                           (70-161)                                     N.sup.ε iPrLys.sup.12,21                                              ______________________________________                                         #Two different reference preparations were used for RIA.                      NIADDKrGH-RP-1 for assays with higher control values and NIADDKrGH-RP-2       for assays with lower control values.                                         *Potencies are calculated using GRF(1-29)NH.sub.2 (=1) as standard by         4point assay (95% CI limits in parentheses) either by comparison to poole     GRF(1-29) NH.sub.2 standards or to standard run in the same bioassay.         Potencies without 95% CI's are estimated by single dose comparisons to        pooled standard responses.                                               

EXAMPLE 22

Using the method of Example 19, the following perisopropylated peptidesare prepared:

1) (N-isopropyl)Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH₂.

2) (N-isopropyl)Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH₂.

3) (N-isopropyl)Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Gly-Ala-(N.sup.ε-isopropyl)Lys-Val-Arg-Leu-NH₂.

4) (N-isopropyl)Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-(N.sup.ε-isopropyl)Lys-Val-Arg-Leu-NH₂.

5) (N-isopropyl)Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-(N.sup.ε-isopropyl)Lys-Val-Arg-Leu-NH₂.

6)(N-isopropyl)His-Ala-Asp-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH.

7) (N-isopropyl)Thr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-NH₂.

8) (N-isopropyl)His-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Ile-Leu-Gly-Gln-Leu-Ala-Arg-(N-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-NH₂.

9)(N-isopropyl)His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-(N-isopropyl)Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-NH₂.

10) (N-isopropyl)Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-(N.sup.ε-isopropyl)Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-(N.sup.ε-isopropyl)Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-OH.

EXAMPLE 23 HpGRF(1-29)-Gly-OH

Using the procedure outlined by K. Horiki, et al., in Chemistry Letters,165-168 (1978), Boc-Gly is coupled to the Merrifield chloromethylatedresin using KF in DMF at about 60° C. for 24 hours with stirring.

After deblocking and neutralization, the peptide chain is builtstep-by-step on the resin. Deblocking, neutralization and addition ofeach amino acid is performed in general accordance with the procedureset forth in detail in Rivier, J. J. Amer. Chem. Soc., 96, 2986-2992(1974). All solvents that are used are carefully degassed by spargingwith an inert gas, e.g. helium or nitrogen, to insure the absence ofoxygen that might undesirably oxidize the sulfur of the Met residue.

Deblocking is preferably carried out in accordance with Schedule A whichfollows:

    ______________________________________                                        SCHEDULE A                                                                    Reagent            Mixing time (Min.)                                         ______________________________________                                        1. 60% TFA/2% ethanedithiol                                                                      10                                                         2. 60% TFA/2% ethanedithiol                                                                      15                                                         3. IPA/1% ethanedithiol                                                                          0.5                                                        4. Et.sub.3 N (10%) in CH.sub.2 Cl.sub.2                                                         0.5                                                        5. MeOH            0.5                                                        6. Et.sub.3 N (10%) in CH.sub.2 Cl.sub.2                                                         0.5                                                        7. MeOH (twice)    0.5                                                        8. CH.sub.2 Cl.sub.2 (twice)                                                                     0.5                                                        ______________________________________                                    

The couplings are preferably carried out as set out in Schedule B whichfollows:

    ______________________________________                                        SCHEDULE B                                                                    Reagent           Mixing time (Min.)                                          ______________________________________                                         9. DCCl          --                                                          10. Boc-amino acid                                                                              50-90                                                       11. MeOH (twice)  0.5                                                         12. CH.sub.2 Cl.sub.2 (twice)                                                                   0.5                                                         13. Ac.sub.2 O (3M) in CH.sub.2 Cl.sub.2                                                        15.0                                                        14. CH.sub.2 CL.sub.2                                                                           0.5                                                         15. MeOH          0.5                                                         16. CH.sub.2 Cl.sub.2 (twice)                                                                   0.5                                                         ______________________________________                                    

Briefly, one to two mmol. of BOC-protected amino acid in methylenechloride is used per gram of resin, plus one equivalent of 1.0 molarDCCI in methylene chloride for two hours. When Boc-Arg(TOS) is beingcoupled, a mixture of 50% DMF and methylene chloride is used. Bzl etheris used as the hydroxyl side-chain protecting group for Ser and Thr.p-Nitrophenyl ester(ONp) may also be used to activate the carboxyl endof Asn or Gln, and for example, Boc-Asn(ONp) can be coupled overnightusing one equivalent of HOBt in a 50% mixture of DMF and methylenechloride, in which case no DCC is added. 2-chloro-benzyloxycarbonyl(Cl-Z is used as the protecting group for the Lys side chain. Tos isused to protect the guanidino group of Arg and the imidazole nitrogen ofHis, and the Glu or Asp side-chain carboxyl group is protected withOBzl. The following blocked amino acids are then successively coupled:Boc-N^(G) -tosyl-Arg, Boc-O-Bzl-Ser, Boc-Met, Boc-Ile, Boc-benzyl-Asp,Boc-Gln, Boc-Leu, Boc-Leu, Boc-N.sup.ε -2-chlorocarbobenzoxy(Cl-Z)-Lys,Boc tosyl-Arg, Boc-Ala, Boc-O-Bzl-Ser, Boc-Gln, Boc-Gly, Boc-Leu,Boc-Val, Boc-Cl-Z-Lys, Boc-tosyl-Arg, Boc-Br-Z-Tyr, Boc-O-Bzl-Ser,Boc-Asn, Boc-O-Bzl-Thr, Boc-Phe, Boc-Ile, Boc-Ala, Boc-Bzl-Asp, Boc-Alaand Boc-Tyr.

In order to cleave and deprotect the protected peptide-resin, it istreated with 1.5 ml. anisole, 0.5 ml. methylethylsulfide and 15 ml.hydrogen fluoride(HF) per gram of peptide-resin, at -20° C. for one-halfhour and at 0° C. for one-half hour. After elimination of the HF underhigh vacuum, the resin-peptide remainder is washed alternately with drydiethyl ether and chloroform, and the peptide is then extracted withdegassed 2N aqueous acetic acid and separated from the resin byfiltration.

The cleaved and deprotected peptide is then dissolved in 0-5% aceticacid and subjected to purification which may include Sephadex G-50 finegel filtration.

The peptide is then further purified by preparative or semi-preparativeHPLC as described in Rivier et al., Peptides: Structure and BiologicalFunction, (1979) pp 125-8 and Marki et al J. Am. Chem. Soc. 103, 3178(1981). Cartridges fitting Waters Associates prep LC-500 are packed with15-20 μ C₁₈ Silica from Vydac (300A). A gradient of CH₃ CN in TEAP isgenerated by a low pressure Eldex gradient maker, as described inRivier, J., J. Lig. Chromatography 1, 343-367 (I978). Thechromatographic fractions are carefully monitored by HPLC, and only thefractions showing substantial purity are pooled. Desalting of thepurified fractions, independently checked for purity, is achieved usinga gradient of CH₃ CN in 0.1% TFA. The center cut is then lyophilized toyield the desired peptide, the purity of which can be greater than 98%.

EXAMPLE 24 [N .sup.α -Isopropyl-Tyr¹, N .sup.ε -isopropyl-Lys¹²,21]GRF(1-40)OH

hGRF(1-40)OH (5mg, 1 μmol) is dissolved in DMF/1% AcoH (approx. 1 mL)and acetone (0.1 mL) Na BH₃ CN (0.5mg) is than added and the mixturestirred for 5 hours. Excess reagent is quenched by adding 4 mL of 10%HOAc/H₂ O followed by 10 mL of H₂ O. The resulting solution islyophilized and the crude product is purified by chromatagraphy on aG-10 sephedex column to yield 4.5mg of product as a white fluffy solid.

The product is found to be homogenous by hplc and gives a negativekaiser test. Fast atom bombardment mass spectrometry confirms thecomposition of the product.

EXAMPLE 25 Evaluation of peptides in vivo in sheep

The purpose of this experiment is to compare the GH releasing activitiesof several GRF analogs selected for expected high potency and the lackof either a terminal amide or a D-amino acid. The analogs to be comparedin wether lambs at 100 ng/kg (IV) are tri-isopropyl-GRF(1-29)-NH₂,tri-isopropyl-GRF(1-29)-OH, tri-isopropyl-GRF(1-40)OH, D-ala², arg¹²,21-GRF (1-29)-OH, D-ala², arg¹²,21 -GRF(1-29)-NH₂, GRF(1-40)-OH andGRF(1-29)-NH₂

A balanced 8×8 Latin square design will be used to test the following 8treatments:

    ______________________________________                                        Group    Treatment            Dose (ng/kg)                                    ______________________________________                                        A        Control, vehicle      0.0                                            B        GRF(1-29)-NH.sub.2   100.0                                           C        GRF(1-40)-OH         100.0                                           D        D-ala.sup.2, arg.sup.12,21 -GRF(1-29)-NH.sub.2                                                     100.0                                           E        D-ala.sup.2, arg.sup.12,21 -GRF(1-29)-OH                                                           100.0                                           F        tri-isopropyl-GRF(1-29)-NH.sub.2                                                                   100.0                                           G        tri-isopropyl-GRF(1-29)-OH                                                                         100.0                                           H        tri-isopropyl-GRF(1-40)-OH                                                                         100.0                                           ______________________________________                                    

The diet used in these evaluations is a standard sheep diet (pelleted)and fresh water ad libitum All animals are fed one daily.

The animals are individually identified by numbered ear tags and held innumbered metabolism cages.

Sheep are maintained in individual metabolism cages at all times. Eachsheep has previously received an indwelling jugular cannula which isflushed daily with heparinized (400-800 IU/ml) sterile saline to helpmaintain potency.

All compounds are solubilized at 5 ug/ml in sterile saline containing 1%Tween 80 and injected intravenously at 100 ng/kg. Control lambs receiveequal volumes of the vehicle only. The drugs are prepared each day justprior to injection to minimize sticking to the vials.

Blood is collected in heparinized tubes at 15 minute intervals for onehour prior to and for two hours after the treatments are administered.Plasma samples are removed by centrifugation and frozen.

Body weights of each animal is taken at the start of the experiment andonce during treatment.

Blood samples are taken from each animal at 15 minute intervals for 3hours each day during the 8 day trial period. The samples are analyzedfor plasma growth hormone (ng/ml). Data obtained are reported below asnear values obtained for each treatment.

    ______________________________________                                        EFFECTS OF VARIOUS GRF ANALOGS ON PLASMA GH                                   LEVELS IN LAMBS INJECTED INTRAVENOUSLY AT 100                                 NG/KG.                                                                                       Plasma GH (ng/ml).sup.a                                                         Pre-inj                                                      GRF Analog       Mean.sup.b                                                                            Post-inj Mean.sup.c                                                                       Peak                                     ______________________________________                                        Control          8.4     8.7         12.2                                     GRF(1-29)-NH.sub.2                                                                             8.9     11.3        23.2                                     GRF(1-40)-OH     9.4     25.5        92.3                                     D-ala.sup.2,arg.sup.12,21 -GRF(1-29)-NH.sub.2                                                  8.9     19.1        62.8                                     D-ala.sup.2,arg.sup.12,21 -GRF(1-29)-OH                                                        8.5     19.4        65.0                                     tri-isopropyl-GRF(1-29)-NH.sub.2                                                               9.7     22.0        71.9                                     tri-isopropyl-GRF(1-29)-OH                                                                     9.4     17.2        53.1                                     tri-isopropyl-GRF(1-40)-OH                                                                     9.5     34.0        108.6                                    ______________________________________                                         .sup.a Means within columns without a common superscript are different (P     < .05).                                                                       .sup.b Means of 5 samples/animal from 8 animals.                              .sup.c Means of 8 samples/animal from 8 animals.                         

What is claimed is:
 1. A peptide having a formula:R₁-A-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-R-Val-Leu-Gly-Gln-Leu-Ser-Ala-R-R-Leu-Leu-Gln-Asp-Ile-I-Ser-R-NH₂,whereinR₁ is N.sup.α -R₁₈ -Tyr or N.sup.α -R₁₈ -D-Tyr; A is Ala or D-Ala; R isArg or Lys; I is Met or Nle; R₁₈ is straight or branched C₂ -C₇ alkyl,cyclohexyl or benzyl; or a pharmaceutically acceptable salt thereof. 2.A peptide according to claim 1, ##STR6##
 3. A peptide according to claim1, ##STR7##
 4. A peptide according to claim 1, ##STR8##
 5. A peptideaccording to claim 1, ##STR9##
 6. A peptide according to claim 1,##STR10##
 7. A peptide according to claim 1, ##STR11##
 8. A peptideaccording to claim 1, ##STR12##
 9. A method for increasing the releaseof growth hormone in mammals, said method comprising administering tosaid mammals from 0.000001 to 0.1 mg/kg of mammalian body weight per dayof a peptide having the formula of the peptide of claim
 1. 10. Themethod according to claim 9, wherein said peptide is:[N.sup.α-Ethyl-Tyr¹, Nle²⁷ ]GRF(1-29)NH₂ ; [N.sup.α -Pentyl-Tyr¹ ]GRF(1-29)NH₂ ;[N.sup.α -Benzyl-Tyr¹ ]GRF(1-29)NH₂ ; [N.sup.α -Cyclohexyl-Tyr¹]GRF(1-29)NH₂ ; [N.sup.α -Isopropyl-Tyr¹ ]GRF(1-29)NH₂ ; [N.sup.α-Isopropyl-Tyr¹, D-Ala² ]GRF(1-29)NH₂ or [N.sup.α -Isopropyl-Tyr¹, Nle²⁷]GRF(1-29)NH₂.
 11. A method of stimulating the release of growth hormonein an animal, which comprises administering to said animal an effectiveamount of the peptide of claim 1 or the salt thereof, pharmaceuticallyacceptable.